CN108866191B

CN108866191B - Methylation-based tumor marker STAMP-EP2

Info

Publication number: CN108866191B
Application number: CN201810830855.5A
Authority: CN
Inventors: 李振艳
Original assignee: Shanghai Epiprobe Biotechnology Co Ltd
Current assignee: Shanghai Epiprobe Biotechnology Co Ltd
Priority date: 2018-07-26
Filing date: 2018-07-26
Publication date: 2021-07-09
Anticipated expiration: 2038-07-26
Also published as: EP3828273A1; US12281361B2; WO2020020072A1; JP7258139B2; EP3828273A4; US20210292850A1; JP2021531046A; CN108866191A

Abstract

The invention provides a methylated tumor marker STAMP-EP2 and application thereof, belonging to the field of molecular biology. The invention provides an application of a methylated tumor marker STAMP-EP2 in preparation of a tumor diagnosis reagent. The tumor marker STAMP-EP2 disclosed by the invention is hypermethylated in all tumor types, hypomethylated in corresponding normal tissues and very high in sensitivity and specificity, and the primer for detecting the STAMP-EP2 can be used for preparing a tumor diagnosis kit.

Description

Methylation-modification-based tumor marker STAMP-EP2

Technical Field

The invention belongs to the field of disease diagnostic markers, and more particularly relates to a Methylation-modified Tumor marker STAMP (Specific Tumor Aligned Methylation of Pan-cancer).

Background

The occurrence and development of tumors are a complex, multi-level, multi-factor, dynamic process, and comprise the intricate and complex interactions of various factors such as external environment, genetic variation, epigenetic variation and the like. The external environmental factors comprise carcinogenic factors such as physical, chemical and biological factors and unhealthy living habits, the genetic variation comprises gene mutation, copy number change, chromosome dislocation and the like, and the epigenetic variation mainly comprises factors such as DNA methylation, histone modification, non-coding RNA and the like. In the process of generating and developing tumors, environmental factors, genetic factors and epigenetic factors supplement each other and act together to cause inactivation of a series of cancer suppressor genes and activation of proto-oncogenes, thereby causing tumors. During the development of a tumor, three factors interact with each other though they are involved. However, in the occurrence and early stage of tumors, the three factors have sequential roles, so that the tumor problem still faces many challenges for human beings, although new surgical methods, targeted therapy, immunotherapy and the like have advanced in recent years, the understanding of people on tumors has many misdistricts, and the major problems of tumor metastasis, recurrence, heterogeneity, drug resistance and the like are urgently needed to be solved.

There are many kinds of tumors in human body, most tissues of human body have tumors, and different types of tumors are divided into many subtypes, especially in recent years, the classification of tumors is more refined by benefiting from the development of the field of tumor molecular biology. The treatment regimens are also quite different for different types, stages or molecular subtypes of tumors.

With the deepening of understanding of tumors and the advancement of scientific technology, many novel tumor markers have been discovered and used for clinical diagnosis. Before 1980, tumor markers are mainly cell secretions of some hormones, enzymes, proteins and the like, for example, carcinoembryonic antigen (CEA), alpha fetal Antigen (AFP) and the like can be used as markers of various tumors such as liver cancer, gastric cancer and the like, carbohydrate antigen 125(CA125) can be used as a marker of cervical cancer, Prostate Specific Antigen (PSA) can be used as a marker of prostate cancer, and the tumor markers are still used clinically at present, but the sensitivity and the accuracy of the tumor markers cannot meet the clinical requirements easily.

The liquid biopsy technology is a technology for diagnosing and predicting tumors by taking circulating tumor cells or circulating tumor DNA in blood as a detection target. The technology is still in a starting stage at present, and has a plurality of defects: firstly, the sensitivity and the specificity are not high enough, the tumor has great heterogeneity and contains cell populations of various subtypes, the clinical sample, particularly a blood sample, has very small proportion of tumor DNA, the existing tumor marker is difficult to meet the sensitivity of clinical requirements, and misdiagnosis is easily caused in clinic; secondly, one marker only has good effect on one or a few tumors, and the DNA source in blood is very complex, so the existing tumor marker cannot deal with the problems of complex tumor source, metastasis and the like. Due to the existence of these complex conditions, many DNA methylation tumor markers have difficulty in having uniform use standards when applied to clinics, and the sensitivity and accuracy of the markers are seriously influenced. The human tumors have a plurality of characteristics and commonalities, and if a marker common to different tumors can be found, the method has great significance in the aspects of tumor screening, diagnosis, treatment, curative effect judgment and the like.

Therefore, in the field of tumor diagnosis, the development of a novel tumor marker which is universal, easy to determine and high in accuracy is urgently needed.

Disclosure of Invention

The invention aims to provide a method for detecting tumors by using DNA methylation modification as a tumor marker and utilizing abnormal hypermethylation phenomenon of specific sites in the tumors.

In a first aspect of the invention, there is provided an isolated polynucleotide comprising: (a) 1, or a polynucleotide having a nucleotide sequence set forth in SEQ ID NO; (b) 2, or a polynucleotide having the nucleotide sequence shown in SEQ ID NO; (c) a fragment of any one of the polynucleotides (a) to (b) above, wherein at least 1 modified CpG site (e.g., 2 to 45, more specifically 3, 5, 10, 15, 20, 25, 30, 40) is present; (d) nucleic acid (e.g., polynucleotide having a nucleotide sequence shown by SEQ ID NO:5 or SEQ ID NO: 6) complementary to the polynucleotides or fragments of (a) to (c) above.

In a preferred embodiment, the modification comprises a 5-methylation modification, a 5-hydroxymethylation modification, a 5-aldehyde methylation modification or a 5-carboxymethylation modification.

In a second aspect of the invention there is provided an isolated polynucleotide which has been converted from said polynucleotide to a sequence corresponding to the first aspect wherein the cytosine C of the modified CpG site is unchanged and the unmodified cytosine is converted to a T or U.

In a preferred embodiment, it is converted from a polynucleotide corresponding to the first aspect described above by bisulfite or bisulfite treatment. In another preferred embodiment, the polynucleotide comprises: (e) 3 or 7; (f) 4 or 8; (g) a fragment of any one of the polynucleotides (e) to (f) above, wherein at least 1 modified CpG site (e.g., 2 to 45, more specifically 3, 5, 10, 15, 20, 25, 30, 40) is present.

In a third aspect of the invention, there is provided a use of the polynucleotide of the first or second aspect for preparing a detection reagent or kit for tumor.

In a preferred embodiment, the tumor includes (but is not limited to): hematological tumors such as leukemia, lymphoma, multiple myeloma; tumors of the digestive system such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer and gallbladder cancer; tumors of the respiratory system such as lung cancer, pleural tumors; tumors of the nervous system such as gliomas, neuroblastoma, meningiomas; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; urinary system tumors such as renal cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer.

In another preferred embodiment, the sample of the tumor comprises: tissue samples, paraffin-embedded samples, blood samples, pleural effusion samples, and alveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, stool samples, urine samples, saliva samples, sputum samples, cerebrospinal fluid samples, cytology smear samples, cervical scraping or swabbing samples, tissue and cell biopsy samples.

In a fourth aspect of the present invention, there is provided a method for preparing a tumor detection reagent, the method comprising: providing the polynucleotide of the first or second aspect, using the full length or fragment of the polynucleotide as a target sequence, and designing a detection reagent for specifically detecting the CpG site modification of the target sequence; wherein, at least 1 (such as 2-45, more specifically 3, 5, 10, 15, 20, 25, 30, 40) modified CpG sites exist in the target sequence; preferably, the detection reagent includes (but is not limited to): a primer and a probe.

In a fifth aspect of the invention, there is provided an agent or a combination of agents which specifically detects CpG site modifications in a target sequence which is a full length or fragment of a polynucleotide according to any one of the first or second aspects, wherein there are at least 1 (e.g. 2 to 45, more particularly 3, 5, 10, 15, 20, 25, 30, 40) modified CpG sites.

In a preferred embodiment, the agent or agents of the combination are directed against a gene sequence (designed based on the gene sequence) comprising the target sequence, the gene sequence comprising a gene Panel or gene group.

In another preferred embodiment, the detection reagent comprises: a primer and a probe.

In another preferred embodiment, the primers are: primers shown in

SEQ ID NOS

5 and 6.

In a sixth aspect of the invention there is provided the use of an agent or a combination of agents according to the fifth aspect of the invention for the manufacture of a kit for the detection of tumours; preferably, the tumor includes (but is not limited to): tumors of the digestive system such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer and gallbladder cancer; tumors of the respiratory system such as lung cancer, pleural tumors; hematological tumors such as leukemia, lymphoma, multiple myeloma; gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; tumors of the nervous system such as gliomas, neuroblastoma, meningiomas; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; urinary system tumors such as renal cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer.

In a seventh aspect of the present invention, there is provided a detection kit comprising: a container, and a reagent or combination of reagents as described previously located in the container; preferably, each reagent is located in a separate container.

In another preferred embodiment, the kit further comprises: bisulfite or bisulfite, DNA purification reagents, DNA extraction reagents, PCR amplification reagents, and/or instructions for use (indicating detection procedures and criteria for determining results).

In an eighth aspect of the present invention, there is provided a method for in vitro detecting a methylation pattern of a sample, comprising: (i) providing a sample, and extracting nucleic acid; (ii) (ii) detecting CpG site modification of a target sequence in the nucleic acid of (i), said target sequence being a polynucleotide of the first aspect as hereinbefore described or a polynucleotide of the second aspect as hereinbefore described converted therefrom.

In a preferred embodiment, in step (3), the method for analyzing comprises: pyrosequencing method, bisulfite conversion sequencing method, methylation chip method, qPCR method, digital PCR method, second-generation sequencing method, third-generation sequencing method, whole genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, MassArray, methylation specific PCR, or their combination, and combined gene group in vitro detection method and in vivo tracing detection method of partial or all methylation sites in the sequence shown in SEQ ID NO. 1. Also, other methylation detection methods and methylation detection methods newly developed in the future can be applied to the present invention.

In another preferred example, step (ii) includes: (1) (ii) treating the product of (i) to convert unmodified cytosine therein to uracil; preferably, the modification comprises a 5-methylation modification, a 5-hydroxymethylation modification, a 5-aldehyde methylation modification or a 5-carboxymethylation modification; preferably, the nucleic acid of step (i) is treated with bisulfite or bisbisulfite; (2) analyzing the CpG sites of the target sequence in the nucleic acid treated in the step (1).

In another preferred embodiment, the methylation pattern abnormality refers to the occurrence of hypermethylation of C in CpG of the polynucleotide.

In another preferred embodiment, the methylation profile method is not aimed at obtaining a diagnosis of the disease directly, or is not a diagnostic method.

In the ninth aspect of the invention, a tumor diagnosis kit is provided, which comprises a primer pair designed by using the sequence shown in the first aspect or the second aspect of the invention and a gene Panel or a gene group containing the sequence, and the characteristics of normal cells and tumor cells are obtained through DNA methylation state detection.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, 20 clinical samples of paracancer-lung cancer were obtained, the paracancer sample was used as a lung cancer control group, the lung cancer sample was used as a lung cancer experimental group, and the methylation values (left panel) and detection specificity and sensitivity (right panel) of STAMP-EP2 in the control group and the experimental group were compared.

FIG. 2, clinically, 10 colorectal cancer parasamples were obtained as a control group, 30 colorectal cancer samples were obtained as an experimental group, and the methylation values (left panel) and detection specificity and sensitivity (right panel) of STAMP-EP2 in the colorectal cancer control group and the experimental group were compared.

Fig. 3 shows that 10 samples of normal stomach (or gastritis) were obtained clinically as a control group, 10 samples of gastric cancer were obtained as an

experimental group

1, 20 samples of gastric cancer were obtained as an experimental group 2, and the methylation values of STAMP-EP2 in the three groups of samples were compared.

FIG. 4, clinically, 10 colorectal cancer parasamples were obtained as a control group, 30 colorectal cancer samples were obtained as an experimental group, and the methylation values (left panel) and detection specificity and sensitivity (right panel) of STAMP-EP2 in the control group and the experimental group were compared.

FIG. 5 shows that 10 normal stomach (or gastritis) samples were obtained clinically as a control group, 10 stomach cancer surgical resection samples as an

experimental group

1, 20 stomach cancer samples as an experimental group 2, and the control group and the experimental group were compared in terms of STAMP-EP2 methylation value (left panel) and detection specificity and sensitivity (right panel).

FIG. 6, 20 pairs of paracancerous-breast cancer samples were obtained clinically, the paracancerous sample was used as a breast cancer control group, the breast cancer sample was used as a breast cancer experimental group, and the methylation values (left panel) and detection specificity and sensitivity (right panel) of STAMP-EP2 in the control group and the experimental group were compared.

FIG. 7, 18 pairs of clinically obtained paracancerous-pancreatic cancer samples, the paracancerous sample as a pancreatic cancer control group, the pancreatic cancer sample as a pancreatic cancer experimental group, and the control group and the experimental group comparing their STAMP-EP2 methylation values (left panel) and detection specificity and sensitivity (right panel).

Fig. 8, 11 clinical samples of paracarcinoma-head and neck cancer including 5 laryngeal carcinoma, 2 tonsil cancer, 2 epiglottic carcinoma, 1 tongue root cancer, and 1 hypopharyngeal carcinoma were obtained, the paracarcinoma samples were used as controls, the cancer tissues were used as experimental groups, and the methylation values of STAMP-EP2 in the head and neck cancer control group and the experimental group were compared.

FIG. 9 is a graph of the clinical acquisition of bile samples of 10 patients with non-cancer, bile samples of 10 patients with gallbladder cancer, a control group with non-cancer, an experimental group with gallbladder cancer, and the comparison of the methylation value (left graph) and the methylation specificity and sensitivity (right graph) of STAMP-EP2 between the control group and the experimental group.

FIG. 10, clinical collection of 10 non-leukemia bone marrow smear samples as control group, 20 leukemia bone marrow smear samples as experimental group, comparison of leukemia control group and experimental group STAMP-EP2 methylation values (left panel) and detection specificity and sensitivity (right panel).

FIG. 11 shows that 8 renal cancer paracancerous control samples were obtained clinically as a control group, 14 renal cancer samples were obtained as an experimental group, and the methylation values (left panel) and the detection specificity and sensitivity (right panel) of STAMP-EP2 in the renal cancer control group and the experimental group were compared.

FIG. 12 shows that 5 samples of bladder cancer paracoccipital sample and non-cancer urine sample were obtained clinically as a control group, 7 samples of bladder cancer tissue and bladder cancer urine sample were obtained as an experimental group, and the STAMP-EP2 methylation values of the bladder cancer control group and the experimental group were compared.

FIG. 13, 20 normal human plasma samples were collected as a control group, and plasma samples of patients with different tumor types including 10 liver cancer plasma samples, 10 pancreatic cancer plasma samples, 10 lung cancer plasma samples, 10 colorectal cancer plasma samples, and 10 breast cancer plasma samples, and the STAMP-EP2 methylation values of the groups were compared.

FIG. 14, methylation differences of CpG sites in tumor cell lines versus non-tumor cell lines.

Detailed Description

The present inventors have made extensive studies and screening in an effort to study Tumor markers, and have provided a general-purpose DNA methylated Tumor marker STAMP (Specific Tumor Aligned Methylation of Pan-cancer), which is hypomethylated in normal tissues and hypermethylated in Tumor tissues, and which can be used for clinical Tumor detection and as a basis for designing a Tumor diagnostic reagent.

Term(s) for

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in the natural state in the living cell is not isolated or purified, but the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in the natural state.

As used herein, a "sample" or "specimen" includes a substance obtained from any individual or isolated tissue, cell, or body fluid (e.g., plasma) suitable for detection of the methylation state of DNA.

As used herein, "highly methylated" refers to the presence of highly methylated, hydroxymethylated, aldehyde methylated or carboxymethylated modifications of CpG in a gene sequence. For example, in Methylation Specific PCR (MSP) assays, a positive PCR result is obtained in a PCR reaction using methylation specific primers, which indicates that the DNA (gene) region under test is hypermethylated. For example, in the case of real-time quantitative methylation-specific PCR, the determination of hypermethylation status can be based on the relative values of methylation status of control samples thereof to analyze statistical differences.

As used herein, tumors include, but are not limited to: hematological tumors such as leukemia, lymphoma, multiple myeloma; tumors of the digestive system such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer and gallbladder cancer; tumors of the respiratory system such as lung cancer, pleural tumors; tumors of the nervous system such as gliomas, neuroblastoma, meningiomas; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; urinary system tumors such as renal cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer.

Gene marker

The present inventors have conducted extensive and intensive studies to find a target useful for diagnosing tumors, and have identified STAMP-EP2 as a target. The methylation state of the sequence region of STAMP-EP2 gene has significant difference between tumor tissue and non-tumor tissue, and the subject can be determined as a high-risk tumor person as long as the abnormal methylation state (high methylation) of the promoter region of one of the genes is detected. Also, the significant differences that STAMP-EP2 presents between tumor and non-tumor tissues are present in a wide range of tumors, including solid as well as non-solid tumors.

Accordingly, the present invention provides an isolated polynucleotide derived from the human genome having a nucleotide sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5 (reverse complement of SEQ ID NO: 1) or SEQ ID NO:6 (reverse complement of SEQ ID NO: 2) that produces 5-methylcytosine (5mC) at the base C position of a plurality of 5 '-CpG-3's in a tumor cell of a tumor patient. The invention also encompasses fragments of the polynucleotides of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 5 or SEQ ID NO. 6, wherein at least 1 (e.g. 2-45, more particularly 3, 5, 10, 15, 20, 25, 30, 40) methylated CpG sites are present. The polynucleotide or fragment can also be used to design a detection reagent or a detection kit.

In some embodiments of the invention, the fragment of the polynucleotide is, for example: a fragment containing the 247 th to 305 th bases of SEQ ID NO.1 (containing the 17 th to 27 th CpG sites of SEQ ID NO. 1); a fragment containing 249 to 307 bases of SEQ ID NO. 2 (containing 18 to 28 CpG sites of SEQ ID NO. 2). The antisense strand of the above fragment is also useful. These segments are examples of preferred embodiments of the present invention, and other segments may be selected based on the information provided by the present invention.

Furthermore, a gene Panel or gene group comprising the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 5 or SEQ ID NO. 6 or a sequence fragment is also encompassed by the present invention. The characteristics of normal cells and tumor cells can also be obtained by detecting the DNA methylation state aiming at the gene Panel or the gene group.

The polynucleotides described above can be used as key regions in the genome to analyze methylation status by a variety of techniques known in the art. Any technique that can be used to analyze methylation status can be used in the present invention.

The above polynucleotides are treated with bisulfite or bisbisulfite, wherein unmethylated cytosines are converted to uracil, while methylated cytosines remain unchanged.

Accordingly, the present invention also provides a polynucleotide obtained by subjecting the above-mentioned polynucleotide to bisulfite or bisulfite treatment, comprising: 3, 4, 7 or 8. These polynucleotides may also be used in the design of detection reagents or detection kits.

The invention also encompasses fragments of the above polynucleotides or polynucleotides obtained by bisulfite or bisulfite treatment of the antisense strand thereof, and wherein at least 1 methylated CpG site is present.

Detection reagent and kit

Based on the new discovery of the invention, the invention also provides a detection reagent designed based on the polynucleotide sequence, which is used for detecting the methylation pattern of the polynucleotide in a sample in vitro. Detection methods and reagents known in the art for determining genomic sequence and methylation status can be used in the present invention.

Accordingly, the present invention provides a method for preparing a tumor detection reagent, comprising: providing the polynucleotide, using the full length or the fragment of the polynucleotide as a target sequence, and designing a detection reagent for specifically detecting the target sequence; wherein at least 1 methylated CpG site is present in the target sequence.

The detection reagent of the present invention includes but is not limited to: primers, probes, and the like.

Such reagents are, for example, primer pairs, and after the sequence of the polynucleotide is known, the design of primers is known to those skilled in the art, both primers flanking the specific sequence of the target gene to be amplified (including CpG sequences, the complement of CpG therein being the region of the gene for which methylation was the result of the methylation, and the complement of TpG therein being the region of the gene for which demethylation was the result of the methylation). It is understood that, according to the novel findings of the present invention, a plurality of primers or probes or other types of detection reagents can be designed by those skilled in the art for the CpG sites at different positions on the target sequence or the combination thereof, and these should be included in the technical solution of the present invention.

The reagents may also be a combination of reagents (primer combination) comprising more than one set of primers, such that the plurality of polynucleotides described above can be amplified separately.

The invention also provides a kit for in vitro detection of the methylation pattern of a polynucleotide in a sample, the kit comprising: a container, and the primer pair located in the container.

In addition, the kit can also comprise various reagents required by DNA extraction, DNA purification, PCR amplification and the like.

In addition, the kit may further comprise an instruction manual, wherein detection operation steps and result judgment standards are indicated, so as to be convenient for the application of the kit by a person skilled in the art.

Detection method

The methylation profile of a polynucleotide can be determined by known techniques (e.g., Methylation Specific PCR (MSP) or real-time quantitative methylation specific PCR, Methylight), or by other techniques that are still under development and will be developed.

Methods for quantitative methylation-specific PCR (QMSP) can also be used to detect methylation levels. This method is based on a continuous optical monitoring of fluorescent PCR, which is more sensitive than the MSP method. The flux is high and the analysis of the result by an electrophoresis method is avoided.

Other techniques that may be used are: pyrosequencing method, bisulfite conversion sequencing method, qPCR method, next generation sequencing method, whole genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology or HPLC method, and combined gene group detection method. It is to be understood that such techniques, as are known in the art and those that will be developed, may be utilized in conjunction with the present invention based on the new disclosure herein.

As a preferred mode of the present invention, there is also provided a method for in vitro detection of methylation patterns of polynucleotides in a sample. The method is based on the principle that: bisulfite or bisulfite can convert unmethylated cytosine to uracil, to thymine during subsequent PCR amplification, while methylated cytosine remains unchanged; thus, after bisulfite or bisulfite treatment of a polynucleotide, the site of methylation generates a polynucleotide polymorphism (SNP) similar to a C/T. The methylation pattern of the polynucleotide in the detection sample is identified based on the principle, and methylated cytosine and unmethylated cytosine can be effectively distinguished.

The method comprises the following steps: the method comprises the following steps: (a) providing a sample, and extracting genomic DNA; (b) treating the genomic DNA of step (a) with bisulfite or bisbisulfite, whereby unmethylated cytosines in the genomic DNA are converted to uracil; (c) analyzing the genomic DNA treated in step (b) for the presence of methylation pattern abnormalities.

The method of the invention can be used for: (i) detecting a sample of the subject, and analyzing whether the subject has the tumor; (ii) distinguish high risk group of tumor. The method may also be used in situations where the objective is not to obtain a direct diagnosis of the disease.

In the preferred embodiment of the present invention, DNA methylation is detected by PCR amplification and pyrosequencing, but those skilled in the art will appreciate that the method is not limited to this method and that other methods of detecting DNA methylation may be used. In carrying out PCR amplification, the primers used are not limited to those provided in the examples.

After the genomic DNA is treated by bisulfite, unmethylated cytosine is converted into uracil, and then converted into thymine in the subsequent PCR process, so that the sequence complexity of the genome can be reduced, and the difficulty of amplifying a specific target fragment by PCR is increased. Therefore, preferably, nested PCR amplification can be adopted, two pairs of primers on the periphery and the inner periphery are designed, two rounds of PCR amplification reaction are carried out, the amplification product of the first round is used as a template for the second round of amplification, and the efficiency and the specificity of the amplification can be effectively improved. However, it is to be understood that the detection method usable in the present invention is not limited thereto.

Through research and verification aiming at clinical samples, the method has very high accuracy when being used for diagnosing clinical tumors. The invention can be applied to the fields of tumor auxiliary diagnosis, curative effect judgment, prognosis monitoring and the like, and has high clinical application value.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 nucleic acid sequence for detection against STAMP-EP2

The sequence of the STAMP-EP2 tumor marker is shown in SEQ ID NO:1(chr5: 140797163-140797701 (hg19/Human)), wherein the underlined bases are methylated CpG sites, and the numbers below the underlined bases indicate the numbers of the sites.

The sequence of the above sequence after bisulfite treatment (wherein Y represents C or U) is as shown in SEQ ID NO: 3:

provides the sequence of the STAMP-EP2 tumor marker, which is shown in the following SEQ ID NO:2(chr5: 140787504-140788044 (hg19/Human)), wherein the underlined mark base is methylated CpG site, and the underlined number indicates the number of the site;

the sequence of the above sequence after bisulfite treatment (wherein Y represents C or U) is as shown in SEQ ID NO: 4:

the reverse complementary sequence of the nucleotide sequence shown in SEQ ID NO.1 is shown as SEQ ID NO. 5:

CGGTCTATTCGGTCCTTCACAAGTAAGTCCCCGCTCTGCGCGTCTACGCTGAAGTGCAGCTTCTCCGCGCTCACTCGCAGCTCGCGAGCCGACACATCCAGGACACTAAGCCCTAGATCCTTAGCGAGGTTCCCCACCACCGAGCCCTTGGCCAGCTCCTCCGGAATCGAGTAGCGGATCGGCTCACACAGCGTGGGGTAGAACAAAGGCAGCAGCAAAGGAAATAGTACCTGCCGCGGGCCGGCCCGGCGCCTCTGCGCGCAGCTCCCTCCCATCGTTCGCTCGGGTTCTCGCTGGGTCCCCGCTTTTCCAGTTGGAGAAAGTGCACTCTACCGCGCTAAAAGAGCATTCGGCCCAGGACAGGAGGAGTCCCGGGTCTCGGAGCTGGCAATCTGGTGTGCTGGGCAAGGTCTGCGCAGCCGGGAGCCTCTGTGTGGGAGCTGGTTTTCTTTTCTGTTGTTGGCTGCGCAGGGAATCCCAGGCTAGAGGCTGAGGGATCCCCGGCGTCAGCGCTTGCTCTGCACTGGCCGACGGCGGCG

the reverse complementary sequence of the nucleotide sequence shown in SEQ ID NO. 2 is as follows SEQ ID NO. 6:

CGGTCTATTCGGTTCTTCACAAGTAAGTCCCCGCTCTCCGCGTCTACGCTGAAGTGCAGCTTCTCCGCGCTCACTCGCAGCTTGCGAGCCGACACATCCAGGACACTGAGCCCTAGATCCTTAGCGAGGTTCCCCACCACCGAGCCCTTGGCCAGCTCCTCCGGAATCGAGTAGCGGATCGGCTCACTCAGGGTGGGGTAGAACAAAGGCAGCAGCAAAGGAAATAGCACCTGCCGCGGGCCGGCCCGGCGCCTCTGCGCGCAGCTCCCTCCCATCGTTCGCTCGGGTTCTCGCTGGGTCCCCGCTTTTCCAGTTGGAGAAAGTGCACTCTACCGCGCTAAAAGAGCATTCGGCCCAGGACAGGAGGAGTCCCGGGTCTCGGAGCTGGCAATCCGCTGTGCTGGGAAAGGTCTGCGCAGCCGGGAGGCTCTGTGTGGGAGCTGGTTTTCTTCTTTCTGTTGTTGGCTGCGCAGGGAATCCCAGGCCAGAGGCTGACGGATCCCCGGCGTCAGCGCTTGCTCTGCACTGGCCGACAGCGGCG

the above-mentioned SEQ ID NO:5 sequence (wherein Y represents C or U) after bisulfite treatment is as follows SEQ ID NO:7 (wherein Y represents C or U):

YGGTUTATTYGGTUUTTUAUAAGTAAGTUUUYGUTUTGYGYGTUTAYGUTGAAGTGUAGUTTUTUYGYGUTUAUTYGUAGUTYGYGAGUYGAUAUATUUAGGAUAUTAAGUUUTAGATUUTTAGYGAGGTTUUUUAUUAUYGAGUUUTTGGUUAGUTUUTUYGGAATYGAGTAGYGGATYGGUTUAUAUAGYGTGGGGTAGAAUAAAGGUAGUAGUAAAGGAAATAGTAUUTGUYGYGGGUYGGUUYGGYGUUTUTGYGYGUAGUTUUUTUUUATYGTTYGUTYGGGTTUTYGUTGGGTUUUYGUTTTTUUAGTTGGAGAAAGTGUAUTUTAUYGYGUTAAAAGAGUATTYGGUUUAGGAUAGGAGGAGTUUYGGGTUTYGGAGUTGGUAATUTGGTGTGUTGGGUAAGGTUTGYGUAGUYGGGAGUUTUTGTGTGGGAGUTGGTTTTUTTTTUTGTTGTTGGUTGYGUAGGGAATUUUAGGUTAGAGGUTGAGGGATUUUYGGYGTUAGYGUTTGUTUTGUAUTGGUYGAYGGYGGYG

the above-mentioned SEQ ID NO:6 sequence (wherein Y represents C or U) after bisulfite treatment is as follows SEQ ID NO:8 (wherein Y represents C or U):

YGGTUTATTYGGTTUTTUAUAAGTAAGTUUUYGUTUTUYGYGTUTAYGUTGAAGTGUAGUTTUTUYGYGUTUAUTYGUAGUTTGYGAGUYGAUAUATUUAGGAUAUTGAGUUUTAGATUUTTAGYGAGGTTUUUUAUUAUYGAGUUUTTGGUUAGUTUUTUYGGAATYGAGTAGYGGATYGGUTUAUTUAGGGTGGGGTAGAAUAAAGGUAGUAGUAAAGGAAATAGUAUUTGUYGYGGGUYGGUUYGGYGUUTUTGYGYGUAGUTUUUTUUUATYGTTYGUTYGGGTTUTYGUTGGGTUUUYGUTTTTUUAGTTGGAGAAAGTGUAUTUTAUYGYGUTAAAAGAGUATTYGGUUUAGGAUAGGAGGAGTUUYGGGTUTYGGAGUTGGUAATUYGUTGTGUTGGGAAAGGTUTGYGUAGUYGGGAGGUTUTGTGTGGGAGUTGGTTTTUTTUTTTUTGTTGTTGGUTGYGUAGGGAATUUUAGGUUAGAGGUTGAYGGATUUUYGGYGTUAGYGUTTGUTUTGUAUTGGUY GAUAGYGGYG

example 2, STAMP-EP 2: lung cancer-clinical case sample validation-pyrosequencing method

1. Obtaining a clinical sample: clinically obtaining 20 pairs of paracancer-lung cancer samples, wherein the paracancer samples serve as a lung cancer control group, and 20 lung cancer samples serve as a lung cancer experimental group;

DNA extraction: extracting DNA of an experimental group and DNA of a control group respectively; the phenol chloroform extraction method is used for the experiment, but is not limited to the method;

3. bisulfite treatment: treating the extracted DNA sample with bisulfite, and strictly operating according to the steps; in this experiment, the EZ DNA Methylation-Gold Kit from ZYMO Research, cat # D5006 was used, but not limited to this Kit;

4. designing a primer: according to the sequence of STAMP-EP2 SEQ ID NO:1 and SEQ ID NO:2, designing a PCR amplification primer and a pyrosequencing primer, wherein the PCR amplification primer and the pyrosequencing primer have the following characteristics that: 1 and SEQ ID NO:2, the primers designed in the experiment can simultaneously detect the nucleotide sequences shown in SEQ ID NO:1, CpG sites No. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 and SEQ ID NO:2, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. Subsequent detection of SEQ ID NO:1, CpG sites No. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 and SEQ ID NO:2, the methylation values of CpG sites No. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28 are used as representatives of the methylation value of STAMP-EP2, and the PCR primer amplification sequence, the pyrosequencing primer sequence, the pyrosequencing on-machine detection sequence and the detection sites are shown in Table 1;

TABLE 1

PCR amplification and agarose gel electrophoresis: taking a sample treated by bisulfite as a PCR product, carrying out PCR amplification, and identifying the specificity of the PCR amplification by agarose gel electrophoresis of the amplified product, wherein the size of an amplified fragment is 143 bp;

6. pyrosequencing: detection was performed by a Pyro Mark Q96 ID pyrosequencer available from QIAGEN, and the procedure was strictly followed;

STAMP-EP2 methylation value calculation: pyrosequencing can detect the sequence of the target region SEQ ID NO:1, CpG sites No. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 and SEQ ID NO:2, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 CpG sites, and calculating the mean value as the methylation value of STAMP-EP2 in the sample;

8. and (4) analyzing results: comparing the methylation values of the lung cancer control group and the lung cancer experimental group STAMP-EP2, as shown in FIG. 1, the result shows that in the lung cancer clinical sample, the methylation value of STAMP-EP2 in the lung cancer experimental group is obviously increased, P is less than 0.0001, the detection sensitivity is 95%, and the specificity is 100%.

Example 3, STAMP-EP 2: colorectal cancer-clinical sample validation-pyrosequencing method

1. Obtaining a clinical sample: 10 colorectal cancer paracancerous samples are obtained clinically and used as a control group, and 30 colorectal cancer samples are obtained and used as an experimental group;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: comparing the methylation values of the control group and the experimental group of the colorectal cancer with the methylation value of the STAMP-EP2, as shown in FIG. 2, the result shows that in the clinical sample of the colorectal cancer, the methylation value of the STAMP-EP2 in the experimental group of the colorectal cancer is obviously increased, the P is less than 0.0001, the detection sensitivity is 93.33%, and the specificity is 100%.

Example 4, STAMP-EP 2: gastric cancer-clinical sample verification-pyrosequencing method

1. Obtaining a clinical sample: 10 samples of normal stomach (or gastritis) were obtained clinically as a control group, 10 samples of gastric cancer surgical resection were obtained as an

experimental group

1, and 20 samples of gastric cancer were obtained as an experimental group 2;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: comparing the methylation value of STAMP-EP2 in the control group of normal stomach (or gastritis) with that in the three groups of samples of the surgical incision experimental group 1 and the gastric cancer experimental group 2, as shown in FIG. 3, the result shows that in the clinical samples of gastric cancer, the methylation value of STAMP-EP2 in the gastric cancer experimental group 2 is obviously increased, and P is less than 0.0001. Meanwhile, the methylation condition of the experimental group 1 of the surgical resection group is between that of the control group of normal stomach (or gastritis) and that of the experimental group 2 of gastric cancer, which shows that the methylation of STAMP-EP2 begins to change at the position of the surgical resection, and the STAMP-EP2 serving as a gastric cancer marker can be used for gastric cancer detection on one hand and gastric cancer surgical resection judgment on the other hand.

Example 5, STAMP-EP 2: cervical cancer-clinical sample validation-pyrosequencing method

1. Obtaining a clinical sample: clinically acquiring 12 cervical cancer parasamples as a control group and 16 cervical cancer samples as an experimental group;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: comparing the methylation values of the cervical cancer control group and the experimental group of STAMP-EP2, as shown in FIG. 4, the result shows that in the clinical cervical cancer sample, the methylation value of STAMP-EP2 in the cervical cancer experimental group is significantly increased, P is less than 0.0001, the detection sensitivity is 93.33%, and the specificity is 100%.

Example 6, STAMP-EP 2: liver cancer-clinical sample verification-pyrosequencing method

1. Obtaining a clinical sample: obtaining 22 pairs of paracancer-liver cancer samples from clinic, wherein the paracancer samples are used as a liver cancer control group, and the liver cancer samples are used as a liver cancer experimental group;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: comparing the methylation values of the control group and the experimental group of liver cancer STAMP-EP2, as shown in FIG. 5, the results show that in the clinical sample of liver cancer, the methylation value of STAMP-EP2 in the experimental group of liver cancer is obviously increased, P is less than 0.0001, the detection sensitivity is 100%, and the specificity is 100%.

Example 7, STAMP-EP 2: breast cancer-clinical sample validation-pyrosequencing method

1. Obtaining a clinical sample: 20 pairs of paracarcinoma-breast cancer samples are obtained clinically, the paracarcinoma samples are used as a breast cancer control group, and the breast cancer samples are used as a breast cancer experimental group;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: comparing the methylation values of the breast cancer control group and the experimental group of STAMP-EP2, as shown in FIG. 6, the result shows that in the breast cancer clinical sample, the methylation value of STAMP-EP2 in the breast cancer experimental group is significantly increased, P is less than 0.0001, the detection sensitivity is 100%, and the specificity is 100%.

Example 8, STAMP-EP 2: pancreatic cancer-clinical sample validation-pyrosequencing method

1. Obtaining a clinical sample: clinically obtaining 18 pairs of paracarcinoma-pancreatic cancer samples, wherein the paracarcinoma samples serve as a pancreatic cancer control group, and the pancreatic cancer samples serve as a pancreatic cancer experimental group;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: comparing the methylation values of the control group and the experimental group of the pancreatic cancer with the methylation value of the STAMP-EP2, as shown in FIG. 7, the result shows that in the clinical sample of the pancreatic cancer, the methylation value of the STAMP-EP2 in the experimental group of the pancreatic cancer is obviously increased, P is less than 0.0001, the detection sensitivity is 88.9%, and the specificity is 100%.

Example 9, STAMP-EP 2: head and neck cancer-clinical sample validation-pyrosequencing method

1. Obtaining a clinical sample: clinically obtaining 11 pairs of paracarcinoma-head and neck cancer samples, including 5 cases of laryngeal carcinoma, 2 cases of tonsil cancer, 2 cases of epiglottis, 1 case of tongue root cancer and 1 case of hypopharyngeal carcinoma, wherein the paracarcinoma samples serve as controls, and the cancer tissues serve as experimental groups;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: comparing the methylation values of the control group and the experimental group of the head and neck cancer with the methylation value of the STAMP-EP2, as shown in FIG. 8, the result shows that in the clinical sample of the head and neck cancer, the methylation value of the STAMP-EP2 in the experimental group of the head and neck cancer is obviously increased, P is less than 0.0001, the detection sensitivity is 100%, and the specificity is 100%.

Example 10, STAMP-EP 2: gallbladder cancer-clinical sample verification-pyrosequencing method

1. Obtaining a clinical sample: clinically obtaining 10 bile samples of non-cancer patients, 10 bile samples of gallbladder cancer patients, wherein the non-cancer samples are used as a gallbladder cancer control group, and the gallbladder cancer samples are used as a gallbladder cancer experimental group;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: comparing the methylation values of the control group and the experimental group of gallbladder cancer with the methylation value of STAMP-EP2, as shown in FIG. 9, the result shows that in the clinical sample of gallbladder cancer, the methylation value of STAMP-EP2 in the experimental group of gallbladder cancer is obviously increased, P is less than 0.0001, the detection sensitivity is 90%, and the specificity is 100%.

Example 11, STAMP-EP 2: leukemia-clinical sample validation-pyrosequencing method

1. Obtaining a clinical sample: 10 non-leukemia bone marrow smear samples were obtained clinically as a control group, and 20 leukemia bone marrow smear samples were obtained as an experimental group;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: comparing the methylation values of the control group and the experimental group of leukemia with the methylation value of STAMP-EP2, as shown in FIG. 10, the results show that in the clinical sample of leukemia, the methylation value of STAMP-EP2 in the experimental group of leukemia is obviously increased, P is less than 0.0001, the detection sensitivity is 100%, and the specificity is 100%.

Example 12, STAMP-EP 2: renal cancer-clinical sample validation-pyrosequencing method

1. Obtaining a clinical sample: clinically acquiring 8 renal cancer paracancerous control samples as a control group and 14 renal cancer samples as an experimental group;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: comparing the methylation values of the kidney cancer control group and the experimental group of STAMP-EP2, as shown in FIG. 11, the results show that in the clinical sample of kidney cancer, the methylation value of STAMP-EP2 in the kidney cancer experimental group is obviously increased, P is less than 0.0001, the detection sensitivity is 92.86%, and the specificity is 100%.

Example 13, STAMP-EP 2: bladder cancer-clinical sample validation-pyrosequencing method

1. Obtaining a clinical sample: clinically acquiring 5 bladder cancer paracampal samples and non-cancer urine samples as a control group, and acquiring 7 bladder cancer tissue samples and bladder cancer urine samples as an experimental group;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: comparing the methylation values of the control group and the experimental group of bladder cancer with the methylation value of STAMP-EP2, as shown in fig. 12, the results showed that in clinical samples of bladder cancer, the methylation value of STAMP-EP2 was significantly increased in the experimental group of bladder cancer.

Example 14 STAMP-EP2, plasma sample-clinical sample validation

1. In order to demonstrate that the SATMP-C tumor marker can also be detected by a liquid biopsy mode, 20 normal human plasma samples are collected by the inventor as a control group, and plasma samples of patients with different tumor types comprise 10 liver cancer plasma samples, 10 pancreatic cancer plasma samples, 10 lung cancer plasma samples, 10 colorectal cancer plasma samples and 10 breast cancer plasma samples;

2. step 2.3.4.5.6.7 is the same as in example 2;

8. and (4) analyzing results: as shown in fig. 13, the methylation value of STAMP-EP2 was significantly increased in the groups of liver cancer, pancreatic cancer, lung cancer, colorectal cancer and breast cancer, compared to the normal human plasma control.

Example 15, STAMP-EP 2: methylation differences of CpG sites in tumor cell lines and non-tumor cell lines

1. Extracting genomic DNA of the hepatoma cell line HepG2 and the normal hepatoma cell line;

2. treating the extracted HepG2 and normal liver cell line genome DNA with bisulfite respectively to be used as a template for subsequent PCR amplification;

3. amplification primers are designed according to the sequences of SEQ ID NO 1 and SEQ ID NO 2, and the primers are designed by a conventional method for amplification aiming at different sequence regions.

After PCR amplification, detecting the specificity of PCR fragments by 2% agarose gel electrophoresis, cutting gel, recovering target fragments, connecting and inserting a T vector, converting competent escherichia coli, coating a bacterial plate, selecting clone sequencing on the next day, and selecting 10 clones per fragment for Sanger sequencing;

the results are shown in FIG. 14, which shows that the region of SEQ ID NO.1 has an average methylation value of 3.0% in the normal liver cell line and 78.3% in the liver cancer cell line HepG2, and that the liver cancer cell line methylation level is significantly higher than that of the normal liver cell line. The region of SEQ ID NO. 2 had an average methylation value of 1.8% in the normal liver cell line and 78.7% in the liver cancer cell line HepG2, with significantly higher levels of liver cancer cell line methylation than the normal liver cell line.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Yisheng Spectrum Biotech Co., Ltd

<120> methylation-modification-based tumor marker STAMP-EP2

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<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213> Intelligent (Homo sapiens)

<400> 1

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gggattccct gcgcagccaa caacagaaaa gaaaaccagc tcccacacag aggctcccgg 120

ctgcgcagac cttgcccagc acaccagatt gccagctccg agacccggga ctcctcctgt 180

cctgggccga atgctctttt agcgcggtag agtgcacttt ctccaactgg aaaagcgggg 240

acccagcgag aacccgagcg aacgatggga gggagctgcg cgcagaggcg ccgggccggc 300

ccgcggcagg tactatttcc tttgctgctg cctttgttct accccacgct gtgtgagccg 360

atccgctact cgattccgga ggagctggcc aagggctcgg tggtggggaa cctcgctaag 420

gatctagggc ttagtgtcct ggatgtgtcg gctcgcgagc tgcgagtgag cgcggagaag 480

ctgcacttca gcgtagacgc gcagagcggg gacttacttg tgaaggaccg aatagaccg 539

<210> 2

<211> 541

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 2

cgccgctgtc ggccagtgca gagcaagcgc tgacgccggg gatccgtcag cctctggcct 60

gggattccct gcgcagccaa caacagaaag aagaaaacca gctcccacac agagcctccc 120

ggctgcgcag acctttccca gcacagcgga ttgccagctc cgagacccgg gactcctcct 180

gtcctgggcc gaatgctctt ttagcgcggt agagtgcact ttctccaact ggaaaagcgg 240

ggacccagcg agaacccgag cgaacgatgg gagggagctg cgcgcagagg cgccgggccg 300

gcccgcggca ggtgctattt cctttgctgc tgcctttgtt ctaccccacc ctgagtgagc 360

cgatccgcta ctcgattccg gaggagctgg ccaagggctc ggtggtgggg aacctcgcta 420

aggatctagg gctcagtgtc ctggatgtgt cggctcgcaa gctgcgagtg agcgcggaga 480

agctgcactt cagcgtagac gcggagagcg gggacttact tgtgaagaac cgaatagacc 540

g 541

<210> 3

<211> 539

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(539)

<223> seq id no:1 post-bisulfite treatment sequence

<220>

<221> misc_feature

<222> (1)..(539)

<223> y is C or U

<400> 3

yguyguygty gguuagtgua gaguaagygu tgayguyggg gatuuutuag uututaguut 60

gggattuuut gyguaguuaa uaauagaaaa gaaaauuagu tuuuauauag aggutuuygg 120

utgyguagau uttguuuagu auauuagatt guuagutuyg agauuyggga utuutuutgt 180

uutggguyga atgututttt agygyggtag agtguauttt utuuaautgg aaaagygggg 240

auuuagygag aauuygagyg aaygatggga gggagutgyg yguagaggyg uyggguyggu 300

uygygguagg tautatttuu tttgutgutg uutttgttut auuuuaygut gtgtgaguyg 360

atuygutaut ygattuygga ggagutgguu aagggutygg tggtggggaa uutygutaag 420

gatutagggu ttagtgtuut ggatgtgtyg gutygygagu tgygagtgag ygyggagaag 480

utguauttua gygtagaygy guagagyggg gauttauttg tgaaggauyg aatagauyg 539

<210> 4

<211> 541

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(541)

<223> seq id no 2 sequence after bisulfite treatment

<220>

<221> misc_feature

<222> (1)..(541)

<223> y is c or u

<400> 4

yguygutgty gguuagtgua gaguaagygu tgayguyggg gatuygtuag uututgguut 60

gggattuuut gyguaguuaa uaauagaaag aagaaaauua gutuuuauau agaguutuuy 120

ggutgyguag auutttuuua guauagygga ttguuagutu ygagauuygg gautuutuut 180

gtuutggguy gaatgututt ttagygyggt agagtguaut ttutuuaaut ggaaaagygg 240

ggauuuagyg agaauuygag ygaaygatgg gagggagutg ygyguagagg yguyggguyg 300

guuygyggua ggtgutattt uutttgutgu tguutttgtt utauuuuauu utgagtgagu 360

ygatuyguta utygattuyg gaggagutgg uuaaggguty ggtggtgggg aauutyguta 420

aggatutagg gutuagtgtu utggatgtgt yggutyguaa gutgygagtg agygyggaga 480

agutguautt uagygtagay gyggagagyg gggauttaut tgtgaagaau ygaatagauy 540

g 541

<210> 5

<211> 539

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 5

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ttctccgcgc tcactcgcag ctcgcgagcc gacacatcca ggacactaag ccctagatcc 120

ttagcgaggt tccccaccac cgagcccttg gccagctcct ccggaatcga gtagcggatc 180

ggctcacaca gcgtggggta gaacaaaggc agcagcaaag gaaatagtac ctgccgcggg 240

ccggcccggc gcctctgcgc gcagctccct cccatcgttc gctcgggttc tcgctgggtc 300

cccgcttttc cagttggaga aagtgcactc taccgcgcta aaagagcatt cggcccagga 360

caggaggagt cccgggtctc ggagctggca atctggtgtg ctgggcaagg tctgcgcagc 420

cgggagcctc tgtgtgggag ctggttttct tttctgttgt tggctgcgca gggaatccca 480

ggctagaggc tgagggatcc ccggcgtcag cgcttgctct gcactggccg acggcggcg 539

<210> 6

<211> 541

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 6

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ttagcgaggt tccccaccac cgagcccttg gccagctcct ccggaatcga gtagcggatc 180

ggctcactca gggtggggta gaacaaaggc agcagcaaag gaaatagcac ctgccgcggg 240

ccggcccggc gcctctgcgc gcagctccct cccatcgttc gctcgggttc tcgctgggtc 300

cccgcttttc cagttggaga aagtgcactc taccgcgcta aaagagcatt cggcccagga 360

caggaggagt cccgggtctc ggagctggca atccgctgtg ctgggaaagg tctgcgcagc 420

cgggaggctc tgtgtgggag ctggttttct tctttctgtt gttggctgcg cagggaatcc 480

caggccagag gctgacggat ccccggcgtc agcgcttgct ctgcactggc cgacagcggc 540

g 541

<210> 7

<211> 539

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> allele

<222> (1)..(539)

<223> seq id no 5 bisulfite treated sequence

<220>

<221> misc_feature

<222> (1)..(539)

<223> y is c or u

<400> 7

yggtutatty ggtuuttuau aagtaagtuu uygututgyg ygtutaygut gaagtguagu 60

ttutuygygu tuautyguag utygygaguy gauauatuua ggauautaag uuutagatuu 120

ttagygaggt tuuuuauuau ygaguuuttg guuagutuut uyggaatyga gtagyggaty 180

ggutuauaua gygtggggta gaauaaaggu aguaguaaag gaaatagtau utguygyggg 240

uygguuyggy guututgygy guagutuuut uuuatygtty gutygggttu tygutgggtu 300

uuyguttttu uagttggaga aagtguautu tauygyguta aaagaguatt ygguuuagga 360

uaggaggagt uuygggtuty ggagutggua atutggtgtg utggguaagg tutgyguagu 420

ygggaguutu tgtgtgggag utggttttut tttutgttgt tggutgygua gggaatuuua 480

ggutagaggu tgagggatuu uyggygtuag yguttgutut guautgguyg ayggyggyg 539

<210> 8

<211> 541

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(541)

<223> seq id no 6 bisulfite treated sequence

<220>

<221> misc_feature

<222> (1)..(541)

<223> y is c or u

<400> 8

yggtutatty ggttuttuau aagtaagtuu uygututuyg ygtutaygut gaagtguagu 60

ttutuygygu tuautyguag uttgygaguy gauauatuua ggauautgag uuutagatuu 120

ttagygaggt tuuuuauuau ygaguuuttg guuagutuut uyggaatyga gtagyggaty 180

ggutuautua gggtggggta gaauaaaggu aguaguaaag gaaataguau utguygyggg 240

uygguuyggy guututgygy guagutuuut uuuatygtty gutygggttu tygutgggtu 300

uuyguttttu uagttggaga aagtguautu tauygyguta aaagaguatt ygguuuagga 360

uaggaggagt uuygggtuty ggagutggua atuygutgtg utgggaaagg tutgyguagu 420

ygggaggutu tgtgtgggag utggttttut tutttutgtt gttggutgyg uagggaatuu 480

uagguuagag gutgayggat uuuyggygtu agyguttgut utguautggu ygauagyggy 540

g 541

<210> 9

<211> 28

<212> DNA

<213> primers (Primer)

<400> 9

gaatgttttt ttagygyggt agagtgta 28

<210> 10

<211> 26

<212> DNA

<213> primers (Primer)

<400> 10

caacaacaaa aaaaataata cctacc 26

<210> 11

<211> 23

<212> DNA

<213> primers (Primer)

<400> 11

ttaattggaa aagyggggat tta 23

<210> 12

<211> 62

<212> DNA

<213> primers (Primer)

<400> 12

gygagaatty gagygaayga tgggagggag ttgygygtag aggygtyggg tyggttygyg 60

gt 62

Claims

1. Use of an isolated polynucleotide fragment in the preparation of a tumor detection reagent or kit, wherein the polynucleotide fragment is a fragment of bases 247 to 305 in SEQ ID NO: 1 and a fragment of bases 247 to 305 in SEQ ID NO: 2 A fragment of bases 249-307, wherein a modified CpG site exists in the fragment; wherein, the tumor is selected from the group consisting of: lung cancer, colorectal cancer, gastric cancer, cervical cancer, liver cancer, breast cancer, pancreatic cancer, head and neck cancer cancer, gallbladder cancer, leukemia, kidney cancer, bladder cancer.

2. The use according to claim 1, wherein the tumor samples include: paraffin-embedded samples, blood samples, pleural effusion samples, bronchoalveolar lavage fluid samples, ascites and lavage fluid samples, bile samples , stool samples, urine samples, saliva samples, sputum samples, cerebrospinal fluid samples, cell smear samples, cervical smear or brush samples, tissue and cell biopsy samples.

3. The use according to claim 1, wherein the fragment is amplified with primers of the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10.

4. A method for preparing a tumor detection reagent, characterized in that the method comprises: providing a polynucleotide, and the polynucleotide fragment is a fragment of bases 247-305 in SEQ ID NO: 1 and SEQ ID Fragment of bases 249 to 307 in NO: 2; using the polynucleotide as the target sequence, design a detection reagent that specifically detects the modification of the CpG site of the target sequence; wherein, the target sequence contains A modified CpG site; the tumor is selected from: lung cancer, colorectal cancer, gastric cancer, cervical cancer, liver cancer, breast cancer, pancreatic cancer, head and neck cancer, gallbladder cancer, leukemia, kidney cancer, bladder cancer.

5. A reagent or a combined reagent, characterized in that it specifically detects the CpG site modification of a target sequence, and the target sequence is a fragment of bases 247 to 305 in SEQ ID NO: 1 and SEQ ID NO. : A fragment of bases 249 to 307 in 2, in which there is a modified CpG site.

6. The reagent or the combined reagent of claim 5, wherein the reagent or the combined reagent is a primer of the sequence shown in SEQ ID NO: 9 and SEQ ID NO: 10.

7. The reagent or the combined reagent of claim 5, wherein the reagent or the combined reagent further comprises a sequencing primer, which is a primer of the sequence shown in SEQ ID NO: 11.

8. Use of any one of the reagents or the combined reagents of claims 5 to 7, for preparing a kit for detecting tumors; the tumor is selected from: the tumor is selected from: lung cancer, colorectal cancer, gastric cancer, cervical cancer Cancer, liver cancer, breast cancer, pancreatic cancer, head and neck cancer, gallbladder cancer, leukemia, kidney cancer, bladder cancer.

9. A detection kit, characterized in that it comprises:

A container, and the reagent or reagent combination of any one of claims 5 to 7 in a container.

10. A method for in vitro detection of a sample methylation profile, which method is not a diagnostic method, characterized in that, comprising:

(i) provide samples and extract nucleic acids;

(ii) detecting the modification of the CpG site of the target sequence in the nucleic acid of (i), the target sequence is the fragment of bases 247-305 in SEQ ID NO: 1 and the bases 249-305 in SEQ ID NO: 2 Fragment at position 307.

11. The method of claim 10, wherein in step (ii), the method for detecting comprises: pyrosequencing method, bisulfite conversion sequencing method, methylation chip method, qPCR method, digital PCR method, next-generation sequencing, third-generation sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing, HPLC, MassArray, methylation-specific PCR, or a combination thereof and all The in vitro detection method and the in vivo tracking detection method of the combined genome group of part or all of the methylation sites in the target sequence are described.

12. The method of claim 10, wherein step (ii) comprises:

(1) The product of (i) is treated to convert unmodified cytosine to uracil

(2) Analyze the modification of the target sequence described in the nucleic acid treated in (1).

13. The method of claim 12, wherein in (1), the modification comprises 5-methylation modification, 5-hydroxymethylation modification, 5-aldehyde methylation modification or 5-carboxylation Methylation modification.

14. The method of claim 12, wherein in (1), the nucleic acid in step (i) is treated with bisulfite or bisulfite.